Modified Vaccinia Ankara virus variant

ABSTRACT

The present invention provides an attenuated virus, which is derived from Modified Vaccinia Ankara virus and characterized by the loss of its capability to reproductively replicate in human cell lines. It further describes recombinant viruses derived from this virus and the use of the virus, or its recombinants, as a medicament or vaccine. A method is provided for inducing an immune response in individuals who may be immune-compromised. In addition, a method is provided for the administration of a therapeutically effective amount of the virus, or its recombinants, in a vaccinia virus prime/vaccinia virus boost inoculation regimen.

The present invention provides an attenuated virus which is derived fromModified Vaccinia Ankara virus and which is characterized by the loss ofits capability to reproductively replicate in human cell lines. Itfurther describes recombinant viruses derived from this virus and theuse of the virus or its recombinants as a medicament or vaccine.Additionally, a method is provided for inducing an immune response evenin immune-compromised patients, patients with pre-existing immunity tothe vaccine virus, or patients undergoing antiviral therapy.

BACKGROUND OF THE INVENTION

Modified Vaccinia Ankara (MVA) virus is related to vaccinia virus, amember of the genera Orthopoxvirus in the family of Poxyiridae. MVA wasgenerated by 516 serial passages on chicken embryo fibroblasts of theAnkara strain of vaccinia virus (CVA) (for review see Mayr, A., et al.Infection 3, 6-14 [1975]). As a consequence of these long-term passages,the resulting MVA virus deleted about 31 kilobases of its genomicsequence and, therefore, was described as highly host cell restricted toavian cells (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). Itwas shown in a variety of animal models that the resulting MVA wassignificantly avirulent (Mayr, A. & Danner, K. [1978] Dev. Biol. Stand.41: 225-34). Additionally, this MVA strain has been tested in clinicaltrials as a vaccine to immunize against the human smallpox disease (Mayret al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987], Stickl etal., Dtsch. med. Wschr. 99, 2386-2392 [1974]). These studies involvedover 120,000 humans, including high-risk patients, and proved thatcompared to vaccinia based vaccines, MVA had diminished virulence orinfectiousness while it induced a good specific immune response.

In the following decades, MVA was engineered for use as a viral vectorfor recombinant gene expression or as a recombinant vaccine (Sutter, G.et al. [1994], Vaccine 12: 1032-40).

In this respect, it is most astonishing that even though Mayr et al.demonstrated during the 1970s that MVA is highly attenuated andavirulent in humans and mammals, some recently reported observations(Blanchard et al., 1998, J Gen Virol 79, 1159-1167; Carroll & Moss,1997, Virology 238, 198-211; Altenberger, U.S. Pat. No. 5,185,146;Ambrosini et al., 1999, J Neurosci Res 55(5), 569) have shown that MVAis not fully attenuated in mammalian and human cell lines since residualreplication might occur in these cells. It is assumed that the resultsreported in these publications have been obtained with various knownstrains of MVA since the viruses used essentially differ in theirproperties, particularly in their growth behavior in various cell lines.

Growth behavior is recognized as an indicator for virus attenuation.Generally, a virus strain is regarded as attenuated if it has lost itscapacity or only has reduced capacity to reproductively replicate inhost cells. The above-mentioned observation, that MVA is not completelyreplication incompetent in human and mammalian cells, brings intoquestion the absolute safety of known MVA as a human vaccine or a vectorfor recombinant vaccines.

Particularly for a vaccine, as well as for a recombinant vaccine, thebalance between the efficacy and the safety of the vaccine vector virusis extremely important.

OBJECT OF THE INVENTION

Thus, an object of the invention is to provide novel virus strainshaving enhanced safety for the development of safer products, such asvaccines or pharmaceuticals. Moreover, a further object is to provide ameans for improving an existing vaccination regimen.

SUMMARY OF THE INVENTION

The invention inter alia comprises the following, alone or incombination:

A method for inducing an immune response for treating a human patientcomprising:

(a) administering to the patient a priming inoculation of an effectiveamount of a MVA virus, wherein the MVA virus is characterized byreproductive replication in vitro in chicken embryo fibroblasts and bybeing non-replicative in vitro in human cell lines selected from thehuman keratinocyte cell line HaCaT, the human embryo kidney cell line293, the human bone osteosarcoma cell line 143B, and the human cervixadenocarcinoma cell line HeLa; and

(b) administering to the patient a boosting inoculation of an effectiveamount of a MVA virus, such a

method wherein the MVA is MVA-BN as deposited at the European Collectionof Cell Cultures (ECACC), Salisbury (UK) under number V00083008 or aderivative thereof, such a

method wherein the MVA virus comprises a heterologous nucleic acidsequence, such a

method wherein the heterologous nucleic acid sequence is selected from asequence encoding at least one antigen, antigenic epitope, and/or atherapeutic compound, such a

method wherein the antigen or antigenic epitope is derived from a virusselected from the family of Influenza virus, Flavivirus, Paramyxovirus,Hepatitis virus, Cytomegalovirus, Human immunodeficiency virus or fromviruses causing hemorrhagic fever, such a

method wherein the antigen or antigenic epitope comprises nef, such a

method wherein an immune response is directed against HIV, such a

method wherein the MVA virus is administered as a vaccine, such a

method wherein the immune response is directed against an orthopoxvirus, selected from smallpox, such a

method wherein the patient is immune compromised, such a

method wherein the patient exhibits preexisting immunity to MVA, such a

method wherein the MVA is administered by intravenous, intramuscular,and/or subcutaneous injection, such a

method wherein the MVA prime/MVA boost inoculation regime induces atleast the same level of immune response when compared to aDNA-prime/vaccinia virus boost inoculation regime, such a

method wherein the MVA is administered at a dose of at least about 1×10⁸TCID₅₀, such a

method wherein the priming inoculation MVA is administered about 4 weeksprior to the boosting inoculation MVA, such a

method for inducing an immune response for treating a mammal comprising:

(a) administering to the mammal a priming inoculation of an effectiveamount of a MVA virus, wherein the MVA virus is characterized byreproductive replication in vitro in chicken embryo fibroblasts and bybeing non-replicative in vitro in human cell lines selected from thehuman keratinocyte cell line HaCaT, the human embryo kidney cell line293, the human bone osteosarcoma cell line 143B, and the human cervixadenocarcinoma cell line HeLa; and

(b) administering to the mammal a boosting inoculation of an effectiveamount of a MVA virus.

A kit for treating a human patient comprising:

(a) a MVA characterized by reproductive replication in vitro in chickenembryo fibroblasts and by being non-replicative in vitro in human celllines selected from the human keratinocyte cell line HaCaT, the humanembryo kidney cell line 293, the human bone osteosarcoma cell line 143B,and the human cervix adenocarcinoma cell line HeLa; and

(b) instructions to administer the MVA in a priming inoculation and in aboosting inoculation, such a

kit wherein the MVA is MVA-BN as deposited at the European Collection ofCell Cultures (ECACC), Salisbury (UK) under number V00083008 or aderivative thereof, such a

kit wherein the MVA virus comprises a heterologous nucleic acidsequence, such a

kit wherein the heterologous nucleic acid sequence is selected from asequence encoding at least one antigen, antigenic epitope, and/or atherapeutic compound, such a

kit wherein the antigen or antigenic epitope is derived from a virusselected from the family of Influenza virus, Flavivirus, Paramyxovirus,Hepatitis virus, Cytomegalovirus, Human immunodeficiency virus or fromviruses causing hemorrhagic fever, such a

kit wherein the MVA virus is administered as a vaccine, such a

kit wherein the treatment is directed against an orthopox virus,selected from smallpox, such a

kit wherein the treatment is directed to a patient who is immunecompromised, such a

kit wherein the MVA is administered by intravenous, intramuscular,and/or subcutaneous injection.

DETAILED DESCRIPTION OF THE INVENTION

To achieve the foregoing objectives, according to a preferred embodimentof the present invention, new vaccinia viruses are provided which arecapable of reproductive replication in non-human cells and cell lines,especially in chicken embryo fibroblasts (CEF), but not capable ofreproductive replication in a human cell line known to permitreplication with known vaccinia strains.

Known vaccinia strains reproductively replicate in at least some humancell lines, in particular the human keratinocyte cell line HaCaT(Boukamp et al. 1988, J Cell Biol 106(3): 761-71). Replication in theHaCaT cell line is predictive for replication in vivo, in particular forin vivo replication in humans. It is demonstrated in the example sectionthat all known vaccinia strains tested that show a residual reproductivereplication in HaCaT also replicate in vivo. Thus, the inventionpreferably relates to vaccinia viruses that do not reproductivelyreplicate in the human cell line HaCaT. Most preferably, the inventionconcerns vaccinia virus strains that are not capable of reproductivereplication in any of the following human cell lines: human cervixadenocarcinoma cell line HeLa (ATCC No. CCL-2), human embryo kidney cellline 293 (ECACC No. 85120602), human bone osteosarcoma cell line 143B(ECACC No. 91112502) and the HaCaT cell line.

The growth behaviour or amplification/replication of a virus is normallyexpressed by the ratio of virus produced from an infected cell (Output)to the amount originally used to infect the cell in the first place(Input) (“amplification ratio”). A ratio of “1” between Output and Inputdefines an amplification status wherein the amount of virus producedfrom the infected cells is the same as the amount initially used toinfect the cells. This ratio is understood to mean that the infectedcells are permissive for virus infection and virus reproduction.

An amplification ratio of less than 1, i.e., a decrease of theamplification below input level, indicates a lack of reproductivereplication and thus, attenuation of the virus. Therefore, it was ofparticular interest for the inventors to identify and isolate a strainthat exhibits an amplification ratio of less than 1 in several humancell lines, in particular all of the human cell lines 143B, HeLa, 293,and HaCaT.

Thus, the term “not capable of reproductive replication” means that thevirus of the present invention exhibits an amplification ratio of lessthan 1 in human cell lines, such as 293 (ECACC No. 85120602), 143B(ECACC No. 91112502), HeLa (ATCC No. CCL-2) and HaCaT (Boukamp et al.1988, J Cell Biol 106(3): 761-71) under the conditions outlined inExample 1 of the present specification. Preferably, the amplificationratio of the virus of the invention is 0.8 or less in each of the abovehuman cell lines, i.e., HeLa, HaCaT, and 143B.

Viruses of the invention are demonstrated in Example 1 and Table 1 notto reproductively replicate in cell lines 143B, HeLa and HaCaT. Theparticular strain of the invention that has been used in the exampleswas deposited on Aug. 30, 2000 at the European Collection of CellCultures (ECACC) under number V00083008. This strain is referred to as“MVA-BN” throughout the Specification. It has already been noted thatthe known MVA strains show residual replication in at least one of thehuman cell lines tested (FIG. 1, Example 1). All known vaccinia strainsshow at least some replication in the cell line HaCaT, whereas the MVAstrains of the invention, in particular MVA-BN, do not reproductivelyreplicate in HaCaT cells. In particular, MVA-BN exhibits anamplification ratio of 0.05 to 0.2 in the human embryo kidney cell line293 (ECACC No. 85120602). In the human bone osteosarcoma cell line 143B(ECACC No. 91112502), the ratio is in the range of 0.0 to 0.6. For thehuman cervix adenocarcinoma cell line HeLa (ATCC No. CCL-2) and thehuman keratinocyte cell line HaCaT (Boukamp et al. 1988, J Cell Biol106(3): 761-71), the amplification ratio is in the range of 0.04 to 0.8and of 0.02 to 0.8, respectively. MVA-BN has an amplification ratio of0.01 to 0.06 in African green monkey kidney cells (CV1: ATCC No.CCL-70). Thus, MVA-BN, which is a representative strain of theinvention, does not reproductively replicate in any of the human celllines tested.

The amplification ratio of MVA-BN is clearly above 1 in chicken embryofibroblasts (CEF: primary cultures). As outlined above, a ratio of morethan “1” indicates reproductive replication since the amount of virusproduced from the infected cells is increased compared to the amount ofvirus that was used to infect the cells. Therefore, the virus can beeasily propagated and amplified in CEF primary cultures with a ratioabove 500.

In a particular embodiment of the present invention, the inventionconcerns derivatives of the virus as deposited under ECACC V0083008.“Derivatives” of the viruses as deposited under ECACC V00083008 refer toviruses exhibiting essentially the same replication characteristics asthe deposited strain but exhibiting differences in one or more parts ofits genome. Viruses having the same “replication characteristics” as thedeposited virus are viruses that replicate with similar amplificationratios as the deposited strain in CEF cells and the cell lines HeLa,HaCaT and 143B; and that show a similar replication in vivo, asdetermined in the AGR129 transgenic mouse model (see below).

In a further preferred embodiment, the vaccinia virus strains of theinvention, in particular MVA-BN and its derivatives, are characterizedby a failure to replicate in vivo. In the context of the presentinvention, “failure to replicate in vivo” refers to viruses that do notreplicate in humans and in the mouse model described below. The “failureto replicate in vivo” can be preferably determined in mice that areincapable of producing mature B and T cells. An example of such mice isthe transgenic mouse model AGR129 (obtained from Mark Sutter, Instituteof Virology, University of Zurich, Zurich, Switzerland). This mousestrain has targeted gene disruptions in the IFN receptor type I(IFN-α/β) and type II (IFN-γ) genes, and in RAG. Due to thesedisruptions, the mice have no IFN system and are incapable of producingmature B and T cells, and as such, are severely immune-compromised andhighly susceptible to a replicating virus. In addition to the AGR129mice, any other mouse strain can be used that is incapable of producingmature B and T cells, and as such, is severely immune-compromised andhighly susceptible to a replicating virus. In particular, the viruses ofthe present invention do not kill AGR129 mice within a time period of atleast 45 days, more preferably within at least 60 days, and mostpreferably within 90 days post infection of the mice with 10⁷ pfu virusadministered via intra-peritoneal injection. Preferably, the virusesthat exhibit “failure to replicate in vivo” are further characterized inthat no virus can be recovered from organs or tissues of the AGR129 mice45 days, preferably 60 days, and most preferably 90 days after infectionof the mice with 10⁷ pfu virus administered via intra-peritonealinjection. Detailed information regarding the infection assays usingAGR129 mice and the assays used to determine whether virus can berecovered from organs and tissues of infected mice can be found in theexample section.

In a further preferred embodiment, the vaccinia virus strains of theinvention, in particular MVA-BN and its derivatives, are characterizedas inducing a higher specific immune response compared to the strainMVA-575, as determined in a lethal challenge mouse model. Details ofthis experiment are outlined in Example 2, shown below. Briefly, in sucha model unvaccinated mice die after infection with replication competentvaccinia strains such as the Western Reserve strain L929 TK+ or IHD-J.Infection with replication competent vaccinia viruses is referred to as“challenge” in the context of description of the lethal challenge model.Four days after the challenge, the mice are usually killed and the viraltitre in the ovaries is determined by standard plaque assays using VEROcells (for more details see example section). The viral titre isdetermined for unvaccinated mice and for mice vaccinated with vaccinaviruses of the present invention. More specifically, the viruses of thepresent invention are characterized in that, in this test after thevaccination with 10² TCID₅₀/ml of virus of the present invention, theovarian virus titres are reduced by at least 70%, preferably by at least80%, and more preferably by at least 90%, compared to unvaccinated mice.

In a further preferred embodiment, the vaccinia viruses of the presentinvention, in particular MVA-BN and its derivatives, are useful forimmunization with prime/boost administration of the vaccine. There havebeen numerous reports suggesting that prime/boost regimes using a knownMVA as a delivery vector induce poor immune responses and are inferiorto DNA-prime/MVA-boost regimes (Schneider et al., 1998, Nat. Med. 4;397-402). In all of those studies the MVA strains that have been usedare different from the vaccinia viruses of the present invention. Toexplain the poor immune response if MVA was used for prime and boostadministration it has been hypothesized that antibodies generated to MVAduring the prime-administration neutralize the MVA administered in thesecond immunization, thereby preventing an effective boost of the immuneresponse. In contrast, DNA-prime/MVA-boost regimes are reported to besuperior at generating high avidity responses because this regimecombines the ability of DNA to effectively prime the immune responsewith the properties of MVA to boost the response in the absence of apre-existing immunity to MVA. Clearly, if a pre-existing immunity to MVAand/or vaccinia prevents boosting of the immune response, then the useof MVA as a vaccine or therapeutic would have limited efficacy,particularly in the individuals that have been previously vaccinatedagainst smallpox. However, according to a further embodiment, thevaccinia virus of the present invention, in particular MVA-BN and itsderivatives, as well as corresponding recombinant viruses harboringheterologous sequences, can be used to efficiently first prime and thenboost immune responses in naive animals, as well as animals with apre-existing immunity to poxviruses. Thus, the vaccinia virus of thepresent invention induces at least substantially the same level ofimmunity in vaccinia virus prime/vaccinia virus boost regimes comparedto DNA-prime/vaccinia virus boost regimes. The term “animal” as used inthe present description is intended to also include human beings. Thus,the virus of the present invention is also useful for prime/boostregimes in human beings. If the virus is a non-recombinant virus such asMVA-BN or a derivative thereof, the virus may be used as a smallpoxvaccine in humans, wherein the same virus can be used in both thepriming and boosting vaccination. If the virus is a recombinant virussuch as MVA-BN or a derivative thereof that encodes a heterologousantigen, the virus may be used in humans as a vaccine against the agentfrom which the heterologous antigen is derived, wherein the same viruscan be used in both the priming and boosting vaccination.

A vaccinia virus is regarded as inducing at least substantially the samelevel of immunity in vaccinia virus prime/vaccinia virus boost regimesif, when compared to DNA-prime/vaccinia virus boost regimes, the CTLresponse, as measured in one of the following two assays (“assay 1” and“assay 2”), preferably in both assays, is at least substantially thesame in vaccinia virus prime/vaccinia virus boost regimes when comparedto DNA-prime/vaccinia virus boost regimes. More preferably, the CTLresponse after vaccinia virus prime/vaccinia virus boost administrationis higher in at least one of the assays, when compared toDNA-prime/vaccinia virus boost regimes. Most preferably, the CTLresponse is higher in both of the following assays.

Assay 1: For vaccinia virus prime/vaccinia virus boost administrations,6-8 week old BALB/c (H-2d) mice are prime-immunized by intravenousadministration with 10⁷ TCID₅₀ vaccinia virus of the inventionexpressing the murine polytope as described in Thomson et al., 1998, J.Immunol. 160, 1717 and then boost-immunized with the same amount of thesame virus, administered in the same manner three weeks later. To thisend, it is necessary to construct a recombinant vaccinia virusexpressing the polytope. Methods to construct such recombinant virusesare known to a person skilled in the art and are described in moredetail below. In DNA prime/vaccinia virus boost regimes the primevaccination is done by intra muscular injection of the mice with 50 μgDNA expressing the same antigen as the vaccinia virus. The boostadministration with the vaccinia virus is done in exactly the same wayas for the vaccinia virus prime/vaccinia virus boost administration. TheDNA plasmid expressing the polytope is also described in the publicationreferenced above, i.e., Thomson, et al. In both regimes, the developmentof a CTL response against the epitopes SYI, RPQ and/or YPH is determinedtwo weeks after the boost administration. The determination of the CTLresponse is preferably done using the ELISPOT analysis as described bySchneider, et al., 1998, Nat. Med. 4, 397-402, and as outlined in theexamples section below for a specific virus of the invention. The virusof the invention is characterized in this experiment in that the CTLimmune response against the epitopes mentioned above, which is inducedby the vaccinia virus prime/vaccinia virus boost administration, issubstantially the same, preferably at least the same, as that induced byDNA prime/vaccinia virus boost administration, as assessed by the numberof IFN-γ producing cells/10⁶ spleen cells (see also experimentalsection).

Assay 2: This assay basically corresponds to assay 1. However, insteadof using 10⁷ TCID₅₀ vaccinia virus administered i.v., as in Assay 1; inAssay 2, 10⁸ TCID₅₀ vaccinia virus of the present invention isadministered by subcutaneous injection for both prime and boostimmunization. The virus of the present invention is characterized inthis experiment in that the CTL immune response against the epitopesmentioned above, which is induced by the vaccinia virus prime/vacciniavirus boost administration, is substantially the same, preferably atleast the same, as that induced by DNA prime/vaccinia virus boostadministration, as assessed by the number of IFN-γ producing cells/10⁶spleen cells (see also experimental section).

The strength of a CTL response as measured in one of the assays shownabove corresponds to the level of protection.

Thus, the viruses of the present invention are particularly suitable forvaccination purposes.

In summary, a representative vaccinia virus of the present invention ischaracterized by having at least one of the following properties:

-   -   (i) capability of reproductive replication in chicken embryo        fibroblasts (CEF), but no capability of reproductive replication        in a human cell line known to permit replication with known        vaccinia strains,    -   (ii) failure to replicate in vivo in those animals, including        humans, in which the virus is used as a vaccine or active        ingredient of a pharmaceutical composition,    -   (iii) induction of a higher specific immune response compared to        a known vaccinia strain and/or    -   (iv) induction of at least substantially the same level of a        specific immune response in vaccinia virus prime/vaccinia virus        boost regimes when compared to DNA-prime/vaccinia virus boost        regimes.

Preferably, the vaccinia virus of the present invention has at least twoof the above properties, and more preferably at least three of the aboveproperties. Most preferred are vaccinia viruses having all of the aboveproperties.

Representative vaccinia virus strains are MVA-575 deposited on Dec. 7,2000 at the European Collection of Animal Cell Cultures (ECACC) with thedeposition number V00120707; and MVA-BN, deposited on Aug. 30, 2000, atECACC with the deposition number V000083008, and derivatives thereof, inparticular if it is intended to vaccinate/treat humans. MVA-BN and itsderivatives are most preferred for humans.

In a further embodiment, the invention concerns a kit for vaccinationcomprising a virus of the present invention for the first vaccination(“priming”) in a first vial/container and for a second vaccination(“boosting”) in a second vial/container. The virus may be anon-recombinant vaccinia virus, i.e., a vaccinia virus that does notcontain heterologous nucleotide sequences. An example of such a vacciniavirus is MVA-BN and its derivatives. Alternatively, the virus may be arecombinant vaccinia virus that contains additional nucleotide sequencesthat are heterologous to the vaccinia virus. As outlined in othersections of the description, the heterologous sequences may code forepitopes that induce a response by the immune system. Thus, it ispossible to use the recombinant vaccinia virus to vaccinate against theproteins or agents comprising the epitope. The viruses may be formulatedas shown below in more detail. The amount of virus that may be used foreach vaccination has been defined above.

A process to obtain a virus of the instant invention may comprise thefollowing steps:

-   -   (i) introducing a vaccinia virus strain, for example MVA-574 or        MVA-575 (ECACC V00120707) into non-human cells in which the        virus is able to reproductively replicate, wherein the non-human        cells are preferably selected from CEF cells,    -   (ii) isolating/enriching virus particles from these cells and    -   (iii) analyzing whether the obtained virus has at least one of        the desired biological properties as previously defined above,        wherein the above steps can optionally be repeated until a virus        with the desired replication characteristics is obtained. The        invention further relates to the viruses obtained by the method        of the instant invention. Methods for determining the expression        of the desired biological properties are explained in other        parts of this description.

In applying this method, a strain of the present invention may beidentified and isolated which corresponds to the strain with theaccession number ECACC V0083008, mentioned above.

The growth behavior of the vaccinia viruses of the present invention, inparticular the growth behavior of MVA-BN, indicates that the strains ofthe present invention are far superior to any other characterized MVAisolates in terms of attenuation in human cell lines and failure toreplicate in vivo. The strains of the present invention are thereforeideal candidates for the development of safer products such as vaccinesor pharmaceuticals, as described below.

In one further embodiment, the virus of the present invention, inparticular MVA-BN and its derivatives, is used as a vaccine againsthuman poxvirus diseases, such as smallpox.

In a further embodiment, the virus of the present invention may berecombinant, i.e., may express heterologous genes as, e.g., antigens orepitopes heterologous to the virus, and may thus be useful as a vaccineto induce an immune response against heterologous antigens or epitopes.

The term “immune response” means the reaction of the immune system whena foreign substance or microorganism enters the organism. By definition,the immune response is divided into a specific and an unspecificreaction although both are closely related. The unspecific immuneresponse is the immediate defence against a wide variety of foreignsubstances and infectious agents. The specific immune response is thedefence raised after a lag phase, when the organism is challenged with asubstance for the first time. The specific immune response is highlyefficient and is responsible for the fact that an individual whorecovers from a specific infection is protected against this specificinfection. Thus, a second infection with the same or a very similarinfectious agent causes much milder symptoms or no symptoms at all,since there is already a “pre-existing immunity” to this agent. Suchimmunity and immunological memory persist for a long time, in some caseseven lifelong. Accordingly, the induction of an immunological memory canbe used for vaccination.

The “immune system” means a complex organ involved in the defence of theorganism against foreign substances and microorganisms. The immunesystem comprises a cellular component, comprising several cell types,such as, e.g., lymphocytes and other cells derived from white bloodcells, and a humoral component, comprising small peptides and complementfactors.

“Vaccination” means that an organism is challenged with an infectiousagent, e.g., an attenuated or inactivated form of the infectious agent,to induce a specific immunity. The term vaccination also covers thechallenge of an organism with recombinant vaccinia viruses of thepresent invention, in particular recombinant MVA-BN and its derivatives,expressing antigens or epitopes that are heterologous to the virus.Examples of such epitopes are provided elsewhere in the description andinclude e.g., epitopes from proteins derived from other viruses, such asthe Dengue virus, Hepatitis C virus, HIV, or epitopes derived fromproteins that are associated with the development of tumors and cancer.Following administration of the recombinant vaccinia virus, the epitopesare expressed and presented to the immune system. A specific immuneresponse against these epitopes may be induced. The organism, thus, isimmunized against the agent/protein containing the epitope that isencoded by the recombinant vaccinia virus.

“Immunity” means partial or complete protection of an organism againstdiseases caused by an infectious agent due to a successful eliminationof a preceding infection with the infectious agent or a characteristicpart thereof. Immunity is based on the existence, induction, andactivation of specialized cells of the immune system.

As indicated above, in one embodiment of the invention the recombinantviruses of the present invention, in particular recombinant MVA-BN andits derivatives, contain at least one heterologous nucleic acidsequence. The term “heterologous” is used hereinafter for anycombination of nucleic acid sequences that is not normally foundintimately associated with the virus in nature; such virus is alsocalled a “recombinant virus”.

According to a further embodiment of the present invention, theheterologous sequences are preferably antigenic epitopes that areselected from any non-vaccinia source. Most preferably, the recombinantvirus expresses one or more antigenic epitopes from: Plasmodiumfalciparum, mycobacteria, influenza virus, viruses of the family offlaviviruses, paramyxoviruses, hepatitis viruses, human immunodeficiencyviruses, or from viruses causing hemorrhagic fever, such as hantavirusesor filoviruses, i.e., ebola or marburg virus.

According to still a further embodiment, but also in addition to theabove-mentioned selection of antigenic epitopes, the heterologoussequences can be selected from another poxyiral or a vaccinia source.These viral sequences can be used to modify the host spectrum or theimmunogenicity of the virus.

In a further embodiment the virus of the present invention may code fora heterologous gene/nucleic acid expressing a therapeutic compound. A“therapeutic compound” encoded by the heterologous nucleic acid in thevirus can be, e.g., a therapeutic nucleic acid, such as an antisensenucleic acid or a peptide or protein with desired biological activity.

According to a further preferred embodiment, the expression of aheterologous nucleic acid sequence is preferably, but not exclusively,under the transcriptional control of a poxvirus promoter, morepreferably of a vaccinia virus promoter.

According to still a further embodiment, the heterologous nucleic acidsequence is preferably inserted into a non-essential region of the virusgenome. In another preferred embodiment of the invention, theheterologous nucleic acid sequence is inserted at a naturally occurringdeletion site of the MVA genome as disclosed in PCT/EP96/02926. Methodsfor inserting heterologous sequences into the poxviral genome are knownto a person skilled in the art.

According to yet another preferred embodiment, the invention alsoincludes the genome of the virus, its recombinants, or functional partsthereof. Such viral sequences can be used to identify or isolate thevirus or its recombinants, e.g., by using PCR, hybridizationtechnologies, or by establishing ELISA assays. Furthermore, such viralsequences can be expressed from an expression vector to produce theencoded protein or peptide that then may supplement deletion mutants ofa virus that lacks the viral sequence contained in the expressionvector.

“Functional part” of the viral genome means a part of the completegenomic sequence that encodes a physical entity, such as a protein,protein domain, or an epitope of a protein. Functional part of the viralgenome also describes parts of the complete genomic sequence that codefor regulatory elements or parts of such elements with individualizedactivity, such as promoter, enhancer, cis- or trans-acting elements.

The recombinant virus of the present invention may be used for theintroduction of a heterologous nucleic acid sequence into a target cell,the sequence being either homologous or heterologous to the target cell.The introduction of a heterologous nucleic acid sequence into a targetcell may be used to produce in vitro heterologous peptides orpolypeptides, and/or complete viruses encoded by the sequence. Thismethod comprises the infection of a host cell with the recombinant MVA;cultivation of the infected host cell under suitable conditions; andisolation and/or enrichment of the peptide, protein and/or virusproduced by the host cell.

Furthermore, the method for introduction of a homologous or heterologoussequence into cells may be applied for in vitro and preferably in vivotherapy. For in vitro therapy, isolated cells that have been previously(ex vivo) infected with the virus are administered to a living animalbody for inducing an immune response. For in vivo therapy, the virus orits recombinants are directly administered to a living animal body toinduce an immune response. In this case, the cells surrounding the siteof inoculation are directly infected in vivo by the virus, or itsrecombinants, of the present invention.

Since the virus of the invention is highly growth restricted in humanand monkey cells and thus, highly attenuated, it is ideal to treat awide range of mammals, including humans. Hence, the present inventionalso provides a pharmaceutical composition and a vaccine, e.g., forinducing an immune response in a living animal body, including a human.The virus of the invention is also safe in any other gene therapyprotocol.

The pharmaceutical composition may generally include one or morepharmaceutical acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchauxiliary substances can be water, saline, glycerol, ethanol, wetting oremulsifying agents, pH buffering substances, or the like. Suitablecarriers are typically large, slowly metabolized molecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, lipid aggregates, or thelike.

For the preparation of vaccines, the virus or a recombinant of thepresent invention, is converted into a physiologically acceptable form.This can be done based on experience in the preparation of poxvirusvaccines used for vaccination against smallpox (as described by Stickl,H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392). For example, thepurified virus is stored at −80° C. with a titre of 5×10⁸ TCID₅₀/mlformulated in about 10 mM Tris, 140 mM NaCl, pH 7.4. For the preparationof vaccine shots, e.g., 10²-10⁸ particles of the virus are lyophilizedin 100 ml of phosphate-buffered saline (PBS) in the presence of 2%peptone and 1% human albumin in an ampoule, preferably a glass ampoule.Alternatively, the vaccine shots can be produced by stepwise,freeze-drying of the virus in a formulation. This formulation cancontain additional additives such as mannitol, dextran, sugar, glycine,lactose, polyvinylpyrrolidone, or other additives, such as antioxidantsor inert gas, stabilizers or recombinant proteins (e.g. human serumalbumin) suitable for in vivo administration. The glass ampoule is thensealed and can be stored between 4° C. and room temperature for severalmonths. However, as long as no need exists the ampoule is storedpreferably at temperatures below −20° C.

For vaccination or therapy, the lyophilisate can be dissolved in 0.1 to0.5 ml of an aqueous solution, preferably physiological saline or Trisbuffer, and administered either systemically or locally, i.e., byparenteral, intramuscular, or any other path of administration know to askilled practitioner. The mode of administration, dose, and number ofadministrations can be optimized by those skilled in the art in a knownmanner.

Additionally according to a further embodiment, the virus of the presentinvention is particularly useful to induce immune responses inimmune-compromised animals, e.g., monkeys (CD4<400/μl of blood) infectedwith SIV, or immune-compromised humans. The term “immune-compromised”describes the status of the immune system of an individual that exhibitsonly incomplete immune responses or has a reduced efficiency in thedefence against infectious agents. Even more interesting and accordingto still a further embodiment, the virus of the present invention canboost immune responses in immune-compromised animals or humans even inthe presence of a pre-existing immunity to poxvirus in these animals orhumans. Of particular interest, the virus of the present invention canalso boost immune responses in animals or humans receiving an antiviral,e.g., antiretroviral therapy. “Antiviral therapy” includes therapeuticconcepts in order to eliminate or suppress viral infection including,e.g., (i) the administration of nucleotide analogs, (ii) theadministration of inhibitors for viral enzymatic activity or viralassembling, or (iii) the administration of cytokines to influence immuneresponses of the host.

According to still a further embodiment, the vaccine is especially, butnot exclusively, applicable in the veterinary field, e.g., immunizationagainst animal pox infection. In small animals, the immunizinginoculation is preferably administered by nasal or parenteraladministration, whereas in larger animals or humans, a subcutaneous,oral, or intramuscular inoculation is preferred.

The inventors have found that a vaccine shot containing an effectivedose of only 10² TCID₅₀ (tissue culture infectious dose) of the virus ofthe present invention is sufficient to induce complete immunity againsta wild type vaccinia virus challenge in mice. This is particularlysurprising since such a high degree of attenuation of the virus of thepresent invention would be expected to negatively influence and thereby,reduce its immunogenicity. Such expectation is based on theunderstanding that for induction of an immune response, the antigenicepitopes must be presented to the immune system in sufficient quantity.A virus that is highly attenuated and thus, not replicating, can onlypresent a very small amount of antigenic epitopes, i.e., as much as thevirus itself incorporates. The amount of antigen carried by viralparticles is not considered to be sufficient for induction of a potentimmune response. However, the virus of the invention stimulates, evenwith a very low effective dose of only 10² TCID₅₀, a potent andprotective immune response in a mouse/vaccinia challenge model. Thus,the virus of the present invention exhibits an unexpected and increasedinduction of specific immunity compared to other characterized MVAstrains. This makes the virus of the present invention and any vaccinederived thereof, especially useful for application in immune-compromisedanimals or humans.

According to still another embodiment of the invention, the virus isused as an adjuvant. An “adjuvant” in the context of the presentdescription refers to an enhancer of the specific immune response invaccines. “Using the virus as adjuvant” means including the virus in apre-existing vaccine to additionally stimulate the immune system of thepatient who receives the vaccine. The immunizing effect of an antigenicepitope in most vaccines is often enhanced by the addition of aso-called adjuvant. An adjuvant co-stimulates the immune system bycausing a stronger specific immune reaction against an antigenic epitopeof a vaccine. This stimulation can be regulated by factors of theunspecific immune system, such as interferon and interleukin. Hence, ina further embodiment of the invention, the virus is used in mammals,including humans, to activate, support, or suppress the immune system,and preferably to activate the immune response against any antigenicdeterminant. The virus may also be used to support the immune system ina situation of increased susceptibility to infection, such as in thecase of stress.

The virus used as an adjuvant may be a non-recombinant virus, i.e., avirus that does not contain heterologous DNA in its genome. An exampleof this type of virus is MVA-BN. Alternatively, the virus used as anadjuvant is a recombinant virus containing in its genome heterologousDNA sequences that are not naturally present in the viral genome. Foruse as an adjuvant, the recombinant viral DNA preferably contains andexpresses genes that code for immune stimulatory peptides or proteinssuch as interleukins.

According to a further embodiment, it is preferred that the virus isinactivated when used as an adjuvant or added to another vaccine. Theinactivation of the virus may be performed by e.g., heat or chemicals,as known in the art. Preferably, the virus is inactivated byβ-propriolacton. According to this embodiment of the invention, theinactivated virus may be added to vaccines against numerous infectiousor proliferative diseases to increase the immune response of the patientto this disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Growth kinetics of different strains of MVA in different celllines. In 1A, the results are grouped according to the MVA strainstested; whereas in 1B, the results are grouped according to the celllines tested. In 1B, the amount of virus recovered from a cell lineafter four days (D4) of culture was determined by plaque assay andexpressed as the ratio of virus recovered after 4 days to the initialinoculum on day 1 (D1).

FIG. 2: Protection provided against a lethal challenge of vacciniafollowing vaccinations with either MVA-BN or MVA-575. The protection ismeasured by the reduction in ovarian titres determined 4 days postchallenge by standard plaque assay.

FIG. 3: Induction of CTL and protection provided against an influenzachallenge using different prime/boost regimes.

3A: Induction of CTL responses to 4 different H-2d restricted epitopesfollowing vaccination with different combinations of DNA or MVA-BNvaccines encoding a murine polytope. BALB/c mice (5 per group) werevaccinated with either DNA (intramuscular) or MVA-BN (subcutaneous) andreceived booster immunizations three weeks later. CTL responses to 4different epitopes encoded by the vaccines (TYQ, influenza; SYI, P.berghei; YPH, cytomegalovirus; RPQ, LCV) were determined using anELISPOT assay 2 weeks post booster immunizations.

3B: Induction of CTL responses to 4 different epitopes followingvaccination with different combinations of DNA or MVA-BN vaccinesencoding a murine polytope. BALB/c mice (5 per group) were vaccinatedwith either DNA (intramuscular) or MVA-BN (intraveneous) and receivedbooster immunizations three weeks later. CTL responses to 4 differentepitopes encoded by the vaccines (TYQ, influenza; SYI, P. berghei; YPH,cytomegalovirus; RPQ, LCV) were determined using an ELISPOT assay 2weeks post booster immunizations.

3C: Frequency of peptide and MVA specific T cells following homologousprime/boost using an optimal dose (1×10⁸ TCID₅₀) of recombinant MVA-BN,administered subcutaneous. Groups of 8 mice were vaccinated with twoshots of the combinations as indicated in the figure. Two weeks afterthe final vaccination, peptide-specific splenocytes were enumeratedusing an IFN-gamma ELISPOT assay. The bars represent the mean number ofspecific spots plus/minus the standard deviation from the mean.

FIG. 4: SIV load of monkeys vaccinated with MVA-BN nef or MVA-BN.

FIG. 5: Survival of vaccinated monkeys following infection with SIV.

FIG. 6: Monkey serum antibody titres to MVA-BN. The antibody titres foreach animal are shown as different shapes, whereas the mean titre isillustrated as a solid rectangle.

FIG. 7: Levels of SIV in immune-compromised monkeys (CD4<400 ml blood)following vaccinations with MVA-BN encoding tat. Monkeys had previouslyreceived three vaccinations with either MVA-BN or MVA-BN nef (week 0, 8,16) and had been infected with a pathogenic isolate of SIV (week 22). Atweek 100, 102 and 106 (indicated by arrows) the monkeys were vaccinatedwith MVA-BN tat.

FIG. 8: SIV levels in monkeys undergoing anti-retroviral therapy andtherapeutic vaccination using MVA-BN. Three groups of monkeys (n=6) wereinfected with SIV and treated daily with PMPA (indicated by black line).At week 10 and 16 the animals were vaccinated (indicated by arrows) witheither mixtures of recombinant MVA or saline.

FIG. 9: Humoral response generated to SIV following infection andvaccination with recombinant MVA. Three groups (n=6) of monkeys wereinfected with a pathogenic isolate of SIV (week 0) and then treated withthe anti-retroviral therapy (PMPA; indicated by bold line). Monkeys werevaccinated with mixtures of recombinant MVA or saline at week 10 and 16.Antibodies to SIV were determined using infected T cell lysates asantigen in a standard ELISA.

FIG. 10: Humoral response generated to MVA in SIV infected monkeysundergoing anti-retroviral therapy. Three groups (n=6) of monkeys wereinfected with a pathogenic isolate of SIV (week 0) and then treated withthe anti-retroviral therapy (PMPA; indicated by bold line). Monkeys werevaccinated with mixtures of recombinant MVA or saline at week 10 and 16.Antibodies to MVA were determined using a standard capture ELISA forMVA.

FIG. 11: Induction of antibodies to MVA following vaccination of micewith different smallpox vaccines. The levels of antibodies generated toMVA following vaccination with MVA-BN (week 0 and 4), was compared toconventional vaccinia strains, Elstree and Wyeth, given via tailscarification (week 0), MVA-572 (week 0 and 4), and MVA-BN and MVA-572given as a pre-Elstree vaccine. MVA-572 has been deposited at theEuropean Collection of Animal Cell Cultures as ECACC V94012707. Thetitres were determined using a capture ELISA and calculated by linearregression using the linear part of the graph and defined as thedilution that resulted in an optical density of 0.3. * MVA-BN:MVA-BN issignificantly (p>0.05) different to MVA-572:MVA-572.

EXAMPLES

The following examples further illustrate the present invention. Itshould be understood by a person skilled in the art that the examplesmay not be interpreted in any way to limit the applicability of thetechnology provided by the present invention to specific application inthese examples.

Example 1 Growth Kinetics of a New Strain of MVA in Selected Cell Linesand Replication in vivo

(1.1) Growth Kinetics in Cell Lines:

To characterize a newly isolated strain of the present invention(further referred to as MVA-BN) the growth kinetics of the new strainwere compared to those of known MVA strains that have already beencharacterized.

The experiment compared the growth kinetics of the following viruses inthe subsequently listed primary cells and cell lines:

-   -   MVA-BN (Virus stock #23, 18. 02. 99 crude, titered at 2.0×10⁷        TCID₅₀/ml);    -   MVA as characterized by Altenburger (U.S. Pat. No. 5,185,146)        and further referred to as MVA-HLR;    -   MVA (passage 575) as characterized by Anton Mayr (Mayr, A., et        al. Infection 3; 6-14) and further referred to as MVA-575 (ECACC        V00120707); and    -   MVA-Vero as characterized in the International Patent        Application PCT/EP01/02703 (WO 01/68820); Virus stock, passage        49, #20, 22.03.99 crude, titered at 4.2×10⁷ TCID₅₀/ml.

The primary cells and cell lines used were:

-   -   CEF Chicken embryo fibroblasts (freshly prepared from SPF eggs);    -   HeLa Human cervix adenocarcinoma (epithelial), ATCC No. CCL-2;    -   143B Human bone osteosarcoma TK-, ECACC No. 91112502;    -   HaCaT Human keratinocyte cell line, Boukamp et al. 1988, J Cell        Biol 106(3): 761-771;    -   BHK Baby hamster kidney, ECACC 85011433;    -   Vero African green monkey kidney fibroblasts, ECACC 85020299;    -   CV1 African green monkey kidney fibroblasts, ECACC 87032605.

For infection the cells were seeded onto 6-well-plates at aconcentration of 5×10⁵ cells/well and incubated overnight at 37° C., 5%CO₂ in DMEM (Gibco, Cat. No. 61965-026) with 2% FCS. The cell culturemedium was removed and cells were infected at approximately moi 0.05 forone hour at 37° C., 5% CO₂ (for infection it is assumed that cellnumbers doubled over night). The amount of virus used for each infectionwas 5×10⁴ TCID₅₀ and is referred to as Input. The cells were then washed3 times with DMEM and finally 1 ml DMEM, 2% FCS was added and the plateswere left to incubate for 96 hours (4 days) at 37° C., 5% CO₂. Theinfections were stopped by freezing the plates at −80° C.; followed bytitration analysis.

Titration Analysis (Immunostaining with a Vaccinia Virus SpecificAntibody)

For titration of amount of virus test cells (CEF) were seeded on96-well-plates in RPMI (Gibco, Cat. No. 61870-010), 7% FCS, 1%Antibiotic/Antimycotic (Gibco, Cat. No. 15240-062) at a concentration of1×10⁴ cells/well and incubated over night at 37° C., 5% CO₂. The6-well-plates containing the infection experiments were frozen/thawed 3times and dilutions of 10⁻¹ to 10⁻¹² were prepared using RPMI growthmedium. Virus dilutions were distributed onto test cells and incubatedfor five days at 37° C., 5% CO₂ to allow CPE (cytopathic effect)development. Test cells were fixed (Acetone/Methanol 1:1) for 10 min,washed with PBS and incubated with polyclonal vaccinia virus specificantibody (Quartett Berlin, Cat. No. 9503-2057) at a 1:1000 dilution inincubation buffer for one hour at RT. After washing twice with PBS(Gibco, Cat. No. 20012-019) the HRP-coupled anti-rabbit antibody(Promega Mannheim, Cat. No. W4011) was added at a 1:1000 dilution inincubation buffer (PBS containing 3% FCS) for one hour at RT. Cells wereagain washed twice with PBS and incubated with staining solution (10 mlPBS+200 μl saturated solution of o-dianisidine in 100% ethanol+15 μlH₂O₂ freshly prepared) until brown spots were visible (two hours).Staining solution was removed and PBS was added to stop the stainingreaction. Every well exhibiting a brown spot was marked as positive forCPE and the titre was calculated using the formula of Kaerber (TCID₅₀based assay) (Kaerber, G. 1931. Arch. Exp. Pathol. Pharmakol. 162, 480).

The viruses were used to infect duplicate sets of cells that wereexpected to be permissive for MVA (i.e., CEF and BHK) and cells expectedto be non-permissive for MVA (i.e., CV-1, Vero, HeLa, 143B and HaCaT).The cells were infected at a low multiplicity of infection, i.e., 0.05infectious units per cell (5×10⁴ TCID₅₀). The virus inoculum was removedand the cells were washed three times to remove any remaining unabsorbedviruses. Infections were left for a total of 4 days when viral extractswere prepared and then titered on CEF cells. Table 1 and FIG. 1 show theresults of the titration assays where values are given as total amountof virus produced after 4 days infection.

It was demonstrated that all viruses amplified well in CEF cells asexpected, since this is a permissive cell line for all MVAs.Additionally, it was demonstrated that all viruses amplified well in BHK(Hamster kidney cell line). MVA-Vero performed the best, since BHK is apermissive cell line for this strain.

Concerning replication in Vero cells (Monkey kidney cell line), MVA-Veroamplified well, as expected, i.e., 1000 fold above Input. MVA-HLR andalso MVA-575 amplified well with a 33-fold and 10-fold increase aboveInput, respectively. Only MVA-BN was found to not amplify as well inthese cells when compared to the other strains, i.e., only a 2-foldincrease above Input.

Also concerning replication in CV1 cells (Monkey kidney cell line), itwas found that MVA-BN is highly attenuated in this cell line. Itexhibited a 200-fold decrease below Input. MVA-575 did not amplify abovethe Input level and also exhibited a slight negative amplification,i.e., 16-fold decrease below Input. MVA-HLR amplified the best with a30-fold increase above Input, followed by MVA-Vero with 5-fold increaseabove Input.

It is most interesting to compare the growth kinetics of the variousviruses in human cell lines. Regarding reproductive replication in 143Bcells (human bone cancer cell line) it was demonstrated that MVA-Verowas the only strain to show amplification above Input (3-fold increase).All other viruses did not amplify above Input, however there was a bigdifference between the MVA-HLR and both MVA-BN and MVA-575. MVA-HLR was“borderline” (1-fold decrease below Input), whereas MVA-BN exhibited thegreatest attenuation (300-fold decrease below Input), followed byMVA-575 (59-fold decrease below Input). To summarize, MVA-BN is superiorwith respect to attenuation in human 143B cells.

Furthermore, concerning replication in HeLa cells (human cervix cancercells) it was demonstrated that MVA-HLR amplified well in this cellline, and even better than it did in the permissive BHK cells(HeLa=125-fold increase above Input; BHK=88-fold increase above Input)MVA-Vero also amplified in this cell line (27-fold increase aboveInput). However, MVA-BN, and also to a lesser extent MVA-575, wereattenuated in these cell lines (MVA-BN=29-fold decrease below Input andMVA-575=6-fold decrease below Input).

Concerning the replication in HaCaT cells (human keratinocyte cellline), it was demonstrated that MVA-HLR amplified well in this cell line(55-fold increase above Input). Both MVA-Vero adapted and MVA-575exhibited amplification in this cell line (1.2 and 1.1-fold increaseabove Input, respectively). However, MVA-BN was the only one todemonstrate attenuation (5-fold decrease below Input).

From this experimental analysis, we may conclude that MVA-BN is the mostattenuated strain in this group of viruses. MVA-BN demonstrates extremeattenuation in human cell lines by exhibiting an amplification ratio of0.05 to 0.2 in human embryo kidney cells (293: ECACC No. 85120602) (datanot incorporated in Table 1). Furthermore, it exhibits an amplificationratio of about 0.0 in 143B cells; an amplification ratio of about 0.04in HeLa cells; and an amplification ratio of about 0.22 in HaCaT cells.Additionally, MVA-BN exhibits an amplification ratio of about 0.0 in CV1cells. Amplification in Vero cells can be observed (ratio of 2.33),however, not to the same extent as in permissive cell lines such as BHKand CEF (compare to Table 1). Thus, MVA-BN is the only MVA strainexhibiting an amplification ratio of less than 1 in each human cell lineexamined, i.e., 143B, Hela, HaCaT, and 293.

MVA-575 exhibits a profile similar to that of MVA-BN, however it is notas attenuated as MVA-BN.

MVA-HLR amplified well in all (human or otherwise) cell lines tested,except for 143B cells. Thus, it can be regarded as replication competentin all cell lines tested, with the exception of 143B cells. In one case,it even amplified better in a human cell line (HeLa) than in apermissive cell line (BHK).

MVA-Vero does exhibit amplification in all cell lines, but to a lesserextent than demonstrated by MVA-HLR (ignoring the 143B result).Nevertheless, it cannot be considered as being in the same “class” withregards to attenuation, as MVA-BN or MVA-575.

1.2 Replication in vivo

Given that some MVA strains clearly replicate in vitro, different MVAstrains were examined with regard to their ability to replicate in vivousing a transgenic mouse model AGR129. This mouse strain has targetedgene disruptions in the IFN receptor type I (IFN-α/β) and type II(IFN-γ) genes, and in RAG. Due to these disruptions, the mice have noIFN system and are incapable of producing mature B and T cells and, assuch, are severely immune-compromised and highly susceptible to areplicating virus. Groups of six mice were immunized (i.p) with 10⁷ pfuof either MVA-BN, MVA-HLR or MVA-572 (used in 120,000 people in Germany)and monitored daily for clinical signs. All mice vaccinated with MVA-HLRor MVA-572 died within 28 and 60 days, respectively. At necropsy, therewere general signs of severe viral infection in the majority of organs.A standard plaque assay measured the recovery of MVA (10⁸ pfu) from theovaries. In contrast, mice vaccinated with the same dose of MVA-BN(corresponding to the deposited strain ECACC V00083008) survived formore than 90 days and no MVA could be recovered from organs or tissues.

When taken together, data from the in vitro and in vivo studies clearlydemonstrate that MVA-BN is more highly attenuated than the parental andcommercial MVA-HLR strain, and may be safe for administration toimmune-compromised subjects.

Example 2 Immunological and in vivo Data in Animal Model Systems

These experiments were designed to compare different dose andvaccination regimens of MVA-BN compared to other MVAs in animal modelsystems.

2.1. Different Strains of MVA Differ in Their Ability to Stimulate theImmune Response.

Replication competent strains of vaccinia induce potent immune responsesin mice and at high doses are lethal. Although MVA are highly attenuatedand have a reduced ability to replicate on mammalian cells, there aredifferences in the attenuation between different strains of MVA. Indeed,MVA-BN appears to be more attenuated than other MVA strains, even theparental strain MVA-575. To determine whether this difference inattenuation affects the efficacy of MVA to induce protective immuneresponses, different doses of MVA-BN and MVA-575 were compared in alethal vaccinia challenge model. The levels of protection were measuredby a reduction in ovarian vaccinia titres determined 4 days postchallenge, as this allowed a quantitative assessment of different dosesand strains of MVA.

Lethal Challenge Model

Specific pathogen-free 6-8-week-old female BALB/c (H-2d mice (n=5) wereimmunized (i.p.) with different doses (10², 10⁴ or 10⁶ TCID₅₀/ml) ofeither MVA-BN or MVA-575. MVA-BN and MVA-575 had been propagated on CEFcells, and had been sucrose purified and formulated in Tris pH 7.4.Three weeks later the mice received a boost of the same dose and strainof MVA, which was followed two weeks later by a lethal challenge (i.p.)with a replication competent strain of vaccinia. As replicationcompetent vaccinia virus (abbreviated as “rVV”) either the strainWR-L929 TK+ or the strain IHD-J were used. Control mice received aplacebo vaccine. The protection was measured by the reduction in ovariantitres determined 4 days post challenge by standard plaque assay. Forthis, the mice were sacrificed on day 4 post the challenge and theovaries were removed, homogenized in PBS (1 ml) and viral titresdetermined by standard plaque assay using VERO cells (Thomson, et al.,1998, J. Immunol. 160: 1717).

Mice vaccinated with two immunizations of either 10⁴ or 10⁶ TCID₅₀/ml ofMVA-BN or MVA-575 were completely protected as judged by a 100%reduction in ovarian rVV titres 4 days post challenge (FIG. 2). Thechallenge virus was cleared. However, differences in the levels ofprotection afforded by MVA-BN or MVA-575 were observed at lower doses.Mice that received two immunizations of 10² TCID₅₀/ml of MVA-575 failedto be protected, as judged by high ovarian rVV titres (mean 3.7×10⁷pfu+/−2.11×10⁷). In contrast, mice vaccinated with the same dose ofMVA-BN exhibited a significant reduction (96%) in ovarian rVV titres(mean 0.21×10⁷ pfu+/−0.287×10⁷). The control mice that received aplacebo vaccine had a mean viral titre of 5.11×10⁷ pfu (+/−3.59×10⁷)(FIG. 2).

Both strains of MVA induce protective immune responses in mice against alethal rVV challenge. Although both strains of MVA are equally efficientat higher doses, differences in their efficacy are clearly evident atsub-optimal doses. MVA-BN is more potent than its parent strain MVA-575at inducing a protective immune response against a lethal rVV challenge,which may be related to the increased attenuation of MVA-BN compared toMVA-575.

2.2. MVA-BN in Prime/Boost Vaccination Regimes

2.2.1.: Induction of Antibodies to MVA Following Vaccination of Micewith Different Smallpox Vaccines

The efficacy of MVA-BN was compared to other MVA and vaccinia strainspreviously used in the eradication of smallpox. These included singleimmunizations using the Elstree and Wyeth vaccinia strains produced inCEF cells and given via tail scarification, and immunizations usingMVA-572 that was previously used in the smallpox eradication program inGermany. In addition, both MVA-BN and MVA-572 were compared as apre-vaccine followed by Elstree via scarification. For each group eightBALB/c mice were used and all MVA vaccinations (1×10⁷ TCID₅₀) were givensubcutaneous at week 0 and week 3. Two weeks following the boostimmunization the mice were challenged with vaccinia (IHD-J) and thetitres in the ovaries were determined 4 days post challenge. Allvaccines and regimes induced 100% protection.

The immune responses induced using these different vaccines or regimeswere measured in animals prior to challenge. Assays to measure levels ofneutralizing antibodies, T cell proliferation, cytokine production(IFN-γ vs IL-4) and IFN-γ production by T cells were used. The level ofthe T cell responses induced by MVA-BN, as measured by ELISPOT, wasgenerally equivalent to other MVA and vaccinia viruses demonstratingbio-equivalence. A weekly analysis of the antibody titres to MVAfollowing the different vaccination regimes revealed that vaccinationswith MVA-BN significantly enhanced the speed and magnitude of theantibody response compared to the other vaccination regimes (FIG. 11).Indeed, the antibody titres to MVA were significantly higher (p>0.05) atweeks 2, 4 and 5 (1 week post boost at week 4) when vaccinated withMVA-BN compared to mice vaccinated with MVA-572. Following the boostvaccination at week 4, the antibody titres were also significantlyhigher in the MVA-BN group compared to the mice receiving a singlevaccination of either the vaccinia strains Elstree or Wyeth. Theseresults clearly demonstrate that 2 vaccinations with MVA-BN induced asuperior antibody response compared to the classical single vaccinationwith traditional vaccinia strains (Elstree and Wyeth) and confirm thefindings from section 1.5 that MVA-BN induces a higher specific immunitythan other MVA strains.

2.2.2.: MVA-Prime and Boost Regimes Generate the Same Level ofProtection as DNA-Prime/MVA-Boost Regimes in an Influenza ChallengeModel.

The efficacy of MVA prime/boost regimes to generate high avidity CTLresponses was assessed and compared to DNA prime/MVA boost regimes thathave been reported to be superior. The different regimes were assessedusing a murine polytope construct encoded by either a DNA vector orMVA-BN and the levels of CTL induction were compared by ELISPOT; whereasthe avidity of the response was measured as the degree of protectionafforded following a challenge with influenza.

Constructs

The DNA plasmid encoding the murine polytope (10 CTL epitopes includinginfluenza, ovalbumin) was described previously (Thomson, et al., 1998,J. Immunol. 160: 1717). This murine polytope was inserted into deletionsite 11 of MVA-BN, propagated on CEF cells, sucrose purified andformulated in Tris pH 7.4.

Vaccination Protocols

In the current study, specific pathogen free 6-8 week old female BALB/c(H-2d) mice were used. Groups of 5 mice were used for ELISPOT analysis,whereas 6 mice per group were used for the influenza challengeexperiments. Mice were vaccinated with different prime/boost regimesusing MVA or DNA encoding the murine polytope, as detailed in theresults. For immunizations with DNA, mice were given a single injectionof 50 μg of endotoxin-free plasmid DNA (in 50 μl of PBS) in thequadricep muscle. Primary immunizations using MVA were done either byintravenous administration of 10⁷ pfu MVA-BN per mouse, or bysubcutaneous administration of 10⁷ pfu or 10⁸ pfu MVA-BN per mouse.Boost immunizations were given three weeks post primary immunization.Boosting with plasmid DNA was done in the same way as the primaryimmunization with DNA (see above). In order to establish CTL responses,standard ELISPOT assays (Schneider et al., 1998, Nat. Med. 4; 397-402)were performed on splenocytes 2 weeks after the last boosterimmunization using the influenza CTL epitope peptide (TYQ), the P.berghei epitope peptide (SYI), the Cytomegalovirus peptide epitope (YPH)and/or the LCV peptide epitope (RPQ).

For the challenge experiments, mice were infected i.n. with a sub-lethaldose of influenza virus, Mem71 (4.5×10⁵ pfu in 50 ml PBS). At day 5post-infection, the lungs were removed and viral titres were determinedin duplicate on Madin-Darby canine kidney cell line using a standardinfluenza plaque assay.

Results:

Using the DNA vaccine alone, the induction of CTL to the 4H-2d epitopesencoded by the murine polytope was poor and only weak responses could bedetected to two of the epitopes for P. Berghei (SYI) and lymphocyticchoriomeningitis virus (RPQ). In contrast, using a DNA prime/MVA boostregime (10⁷ pfu MVA-BN given subcutaneous) there were significantly moreCTL induced to SLY (8-fold increase) and RPQ (3-fold increase) andresponses were also observed to a third epitope for murinecytomegalovirus (YPH) (FIG. 3A). However, 10⁷ pfu MVA-BN givensubcutaneous in a homologous prime/boost regime induced the same levelof response as DNA followed by MVA-BN (FIG. 3A). Surprisingly, there wasno significant difference in the numbers of CTLs induced to the threeepitopes when one immunization of MVA-BN (10⁷ TCID₅₀) was used,indicating that a secondary immunization with MVA-BN did notsignificantly boost CTL responses.

The subcutaneous administration of 10⁷ pfu MVA has previously been shownto be the most inefficient route and virus concentration for vaccinationusing other strains of MVA; particularly when compared to intravenousimmunizations (Schneider, et al. 1998). In order to define optimalimmunization regimes, the above protocol was repeated using variousamounts of virus and modes of administration. In one experiment, 10⁷ pfuMVA-BN was given intravenously (FIG. 3B). In another experiment, 10⁸ pfuMVA-BN was administered subcutaneous (FIG. 3C). In both of theseexperiments, MVA-BN prime/boost immunizations induced higher mean CTLnumbers to all three CTL epitopes when compared to DNA prime/MVA boostregimes. Also unlike 10⁷ pfu MVA-BN administered subcutaneous,immunization with 10⁷ pfu MVA-BN given intravenously and immunizationwith 10⁸ pfu given subcutaneous significantly boosted the CTL response.This clearly indicates that MVA-BN can be used to boost CTL responses inthe presence of a pre-existing immunity to the vector.

2.2.3.: Efficacy of a MVA-BN nef Vaccine in SIV Infected Rhesus Monkeys.

To determine the efficacy of a MVA-BN nef vaccine, the viral load anddelay of disease following a challenge with a virulent primary isolateof SIV were assessed. Another objective of the study was to determinewhether MVA-BN could be used to safely boost immune responses inimmune-compromised monkeys with a pre-existing immunity to MVA.

Vaccination Protocols

Two groups (n=6) of rhesus monkeys (Macaca mulalta) were vaccinated witha bolus intramuscular injection using either MVA-BN alone or arecombinant MVA-BN nef at week 0, 8 and 16. On week 22, all monkeys werechallenged with 50 MID₅₀ of a pathogenic cell-associated SIV stock (1XC)from primary, uncultured rhesus monkey PBMC by the intravenous route.The clinical status of the animals was frequently monitored and regularblood samples were taken for the measurement of viremia, immuneparameters, and a full range of hematology and blood clinical chemistryparameters. Animals that developed AIDs-like disease were sacrificed.The surviving monkeys were monitored for 99 weeks post vaccination. Atweek 100 the surviving monkeys were immunized i.m. with MVA-BN tat andreceived further immunizations with the same MVA-BN tat at week 10² and10⁶.

No adverse effects were observed following any of the vaccinations witheither MVA-BN or MVA-BN nef. Following the infection of the monkeys withSIV, the levels of viremia rose sharply and peaked two weeks postinfection (FIG. 4). Due to the large standard deviations within thegroups, there was no significant difference in the mean levels of SIVbetween the groups vaccinated with MVA-BN nef or MVA-BN. However, therewas a general 10 fold lower SIV load in the group vaccinated with theMVA-BN nef compared to the control (MVA-BN) group. Furthermore, after 35weeks following infection (the initial observation period), only 1 outof the six monkeys vaccinated with MVA-BN nef had to be euthanized dueto the severity of the disease, compared to 4 out of the 6 animals inthe control group (FIG. 5). The development of disease clearlycorrelated with a higher virus load and, as such, the animals wereobserved for an additional 29 weeks post infection. The MVA-BN nefvaccine appeared to delay the progression of the disease compared to thecontrol group, and even at week 46 post-infection 5 out of the 6 MVA-BNnef animals survived (FIG. 5). However, by week 59 post-infection, twoadditional animals in the nef vaccinated group were euthanized leavingfive surviving animals (three from the MVA-BN nef group and twovaccinated with MVA-BN). An examination of the antibody titres generatedto MVA-BN in these 12 monkeys clearly demonstrated that MVA-BN couldboost the immune response even in the presence of a pre-existingimmunity to MVA (FIG. 6). Following the primary immunization with eitherMVA-BN or MVA-BN nef, all monkeys generated an antibody response to MVAwith a mean titre of 1000. This antibody response was significantlyboosted following the secondary immunization, clearly demonstrating thatMVA can be used to prime/boost immune response in healthy monkeys. Theseantibody titres gradually declined, although by week 49post-immunization the titres plateaued, such that the mean titres to MVAat week 99 were 2000.

The five surviving monkeys were SIV infected and immune-compromised withCD4 counts lower than 400/μl blood. To investigate the impact of usingMVA-BN in immune-compromised monkeys the five animals were vaccinationthree times with MVA-BN tat week 100, 102 and 106 post initialvaccination. The first immunization with MVA-BN tat significantlyboosted the antibody response to MVA in the immune-compromised monkeys.The response was further boosted with the third immunization six weekslater (FIG. 6). These results demonstrate that MVA-BN can boost theimmune response in the presence of a significant pre-existing immunityto MVA, even in immune-compromised monkeys. Although the monkeys' immuneresponses were boosted following immunization with MVA-BN tat, thelevels of SIV remained stable. This indicates that immunization withMVA-BN is safe and does not affect SIV levels in immune-compromisedmonkeys (FIG. 7).

This study demonstrated that MVA-BN is able to prime/boost immuneresponses in immune-compromised rhesus monkeys. It also demonstratedthat MVA-BN immunizations are safe and do not affect the levels ofviremia in SIV infected animals. The delay in the progression ofAIDS-like disease in the animals vaccinated with the MVA-BN nef vaccineindicates that an immune response was successfully generated to nef.

2.2.4.: Therapeutic Vaccination of SIV-Infected Monkeys UndergoingAnti-Retroviral Treatment

An MVA-BN based therapeutic HIV vaccine is likely to be used inindividuals undergoing anti-retroviral therapy. Therefore, this studywas designed to investigate the safety (effect on SIV levels) andefficacy of recombinant MVAs encoding a variety of SIV antigens (gag,pol, env, rev, tat, and nef) in SIV infected monkeys treated with PMPA.PMPA is a nucleoside analogue that is effective against HIV and SIV(Rosenwirth, B. et al., 2000, J Virol 74, 1704-11).

Constructs

All the recombinant MVA constructs were propagated on CEF cells, sucrosepurified and formulated in Tris pH 7.4.

Vaccination Protocol

Three groups (n=6) of rhesus monkeys (Macaca mulatta) were infected with50 MID₅₀ of a pathogenic primary SIV isolated (1XC) and then treateddaily with PMPA (60 mg/kg given s.c.) for 19 weeks. At week 10, animalswere vaccinated with recombinant MVA-BN (i.m.), or saline, and receivedidentical vaccinations 6 weeks later. Group 1 received a mixture of MVAgag-pol and MVA-env, group 2 received MVA-tat, MVA-rev and MVA-nef,whereas Group 3 received saline. The clinical status of the animals wasfrequently monitored and regular blood samples were taken for themeasurement of viremia, immune parameters, and a full range ofhematology and blood clinical chemistry parameters.

All animals established high SIV loads that peaked 2 weeks postinfection (FIG. 8). Following daily treatment with PMPA, the SIV levelsdecreased and stabilized to low levels by week 9. As in the previousstudy, vaccinations with MVA at week 10 and 16 had no effect on the SIVlevels, indicating that MVA-BN is a safe vaccine vector forimmune-compromised animals. Once the animals came off PMPA treatment(week 21) the SIV levels increased. Although three animals in Group 1had reduced levels of SIV when compared to control Group 3, there was nosignificant difference in the mean SIV load between any of the groupsfollowing the end of PMPA treatment (FIG. 8). Using an ELISA to SIVinfected T-cell lysates, animals in all groups generated an antibodyresponse to SIV by week 4 following infection (FIG. 9). The SIV antibodytitre in the control group (saline) dropped during the PMPA treatmentand increased rapidly when PMPA treatment stopped, reflecting the dropand subsequent increase in SIV levels during anti-retroviral therapy(FIG. 9). A similar pattern in SIV antibody titre was observed in Group2, which received MVA-tat, MVA-rev and MVA-nef; possibly reflecting theunder-expression of these regulatory proteins in the SIV infected T celllysates used in the ELISA. In contrast however, the anti-SIV antibody sin Group 1 increased following the vaccinations with MVA gag-pol andMVA-env at week 10, indicating that recombinant MVA-BN can boost theimmune response to SIV in (SIV) infected animals undergoinganti-retroviral therapy. Importantly, the anti-SIV antibody titres wereboosted following the secondary immunization at week 16, againdemonstrating that MVA can boost immune responses in immune-compromisedanimals, even in the presence of a pre-existing immunity to MVA (FIG.8). The anti-MVA antibody titres in Group 1 also reflected this patternwith the generation of an antibody response following the primaryimmunization, and this was significantly boosted following the secondaryvaccination (FIG. 10).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference.

TABLE 1 CEF HeLa HaCaT 143B BHK Vero CV-1 MVA- 579.73 0.04 0.22 0.0065.88 2.33 0.00 BN MVA- 796.53 0.15 1.17 0.02 131.22 10.66 0.06 575 MVA-86.68 124.97 59.09 0.83 87.86 34.97 29.70 HLR MVA- 251.89 27.41 1.282.91 702.77 1416.46 4.48 Vero

-   Virus amplification above the input level after 4 days infection-   Amplification ratio=output TCID₅₀−input TCID₅₀.-   Values are in TCID₅₀.

1. An isolated Modified Vaccinia Ankara (MVA) virus characterized byhaving the capability of reproductive replication in vitro in chickenembryo fibroblasts (CEF), but no capability of reproductive replicationin vitro in the human keratinocyte cell line HaCaT and the human cervixadenocarcinoma cell line HeLa: wherein the isolated MVA virus isgenerated by: (a) introducing a Modified Vaccinia Ankara strain intonon-human cells in which the virus is able to reproductively replicate;(b) isolating an MVA virus from the infected cells; and (c) determiningthat said isolated MVA virus has the capability of reproductivereplication in vitro in chicken embryo fibroblasts (CEF), but nocapability of reproductive replication in vitro in the humankeratinocyte cell line HaCaT and the human cervix adenocarcinoma cellline HeLa.
 2. The isolated MVA virus of claim 1, wherein the isolatedMVA virus is generated by introducing the Modified Vaccinia Ankarastrain into CEF cells.
 3. The isolated MVA virus of claim 1, wherein theMVA virus is generated by introducing the Modified Vaccinia Ankarastrain into BHK cells.
 4. The isolated MVA virus of claim 1, wherein theisolated MVA virus is generated by introducing an MVA-572 strain intonon-human cells in which the virus is able to reproductively replicate.5. The isolated MVA virus of claim 1, wherein the isolated MVA virus isgenerated by introducing an MVA-575 strain into non-human cells in whichthe virus is able to reproductively replicate.
 6. The isolated MVA virusof claim 1, wherein the isolated MVA virus comprises a heterologousnucleic acid sequence.
 7. A method for generating a Modified VacciniaAnkara (MVA) virus comprising: (a) introducing a Modified VacciniaAnkara strain into non-human cells in which the virus is able toreproductively replicate; (b) isolating an MVA virus from the infectedcells; and (c) determining that said isolated MVA virus has capabilityof reproductive replication in vitro in chicken embryo fibroblasts(CEF), but no capability of reproductive replication in vitro in thehuman keratinocyte cell line HaCaT and the human cervix adenocarcinomacell line HeLa.
 8. The method of claim 7, comprising introducing theModified Vaccinia Ankara strain into CEF cells.
 9. The method of claim7, comprising introducing the Modified Vaccinia Ankara strain into BHKcells.
 10. The method of claim 7, comprising introducing an MVA-572strain into non-human cells in which the virus is able to reproductivelyreplicate.
 11. The method of claim 7, comprising introducing an MVA-575strain into non-human cells in which the virus is able to reproductivelyreplicate.
 12. An isolated Modified Vaccinia Ankara (MVA) viruscharacterized by having the capability of reproductive replication invitro in chicken embryo fibroblasts (CEF) and by being more attenuatedthan MVA-575 in the human keratinocyte cell line HaCaT, in the humanbone osteosarcoma cell line 143B, and in the human cervix adenocarcinomacell line HeLa.
 13. The isolated MVA virus of claim 12, wherein theisolated MVA virus comprises a heterologous nucleic acid sequence. 14.The isolated MVA virus of claim 12,wherein the isolated MVA virus iscapable of a replication amplification ratio of greater than 500 in CEFcells.
 15. A method for generating a Modified Vaccinia Ankara (MVA)virus comprising: (a) introducing a Modified Vaccinia Ankara strain intonon-human cells in which the virus is able to reproductively replicate;(b) isolating an MVA virus from the infected cells; and (c) determiningthat said isolated MVA virus has the capability of reproductivereplication in vitro in chicken embryo fibroblasts (CEF), and is moreattenuated than MVA-575 in the human keratinocyte cell line HaCaT, inthe human bone osteosarcoma cell line 143B, and in the human cervixadenocarcinoma cell line Hela.
 16. The method of claim 15, comprisingintroducing the Modified Vaccinia Ankara strain into CEF cells.
 17. Themethod of claim 15, comprising introducing the Modified Vaccinia Ankarastrain into BHK cells.
 18. The method of claim 15, comprisingintroducing an MVA572 strain into non-human cells in which the virus isable to reproductively replicate.
 19. The method of claim 15, comprisingintroducing an MVA-575 strain into non-human cells in which the virus isable to reproductively replicate.
 20. A kit comprising the MVA virus ofclaim 1 for a first vaccination in a first vial and for a secondvaccination in a second vial.
 21. A kit comprising the MVA virus ofclaim 12 for first vaccination in a first vial and for a secondvaccination in a second vial.